The present invention relates to medicine and more particularly to pharmacology and therapy, and is intended to be used for preventing and treating various diseases by way of increasing endogenous production of cytokines and hemopoietic factors.
It has been known that a number of endogenously produced mammalian humoral factors, i.e. cytokines and hernopoietic factors possess important biological activities that are considerably helpful in treating various human diseases1,2. Many of these factors are being tested in man, those with proven efficiency being commercially available as medicinal agents.
The following cytokines and hemopoietic factors are being most extensively researched in oncology: interleukin 2 (IL-2)3,4, tumor necrosis factor alpha (TNF-xcex1)5, erythropoietin, macrophage-granulocyte and granulocyte colony-stimulating factors (GM-CSF and G-CSF, respectively6,7). No less actively is being studied the use of cytokines and hemopoietic factors for the treatment of infectious disease: interferons (IFN-xcex3 and IFN-xcex2)8,9,10, colony-stimulating factors11,12, and the like13. Colony-stimulating factors and erythropoietin are broadly used in hematology14,15.
However, the medicinal use of these exogenously administered agents has its limitations associated with the lack of acceptable drug formulations or their exorbitant cost, a short half-life of these substances in biological media, difficulties in dose finding as well as numerous toxic and allergic effects16,17, since even the recombinant products are more or less immunogenic to the human organism because of the processing fluctuations in the course of the artificial synthesis.
In this regard, in view of achieving a more invariable and significant therapeutic effect free of adverse reactions, it is preferable to induce the endogenous production of the autologous cytokines and hemopoietic factors immediately within the organism of a subject. The remedial effect due to such intrinsic stimulation is free of all the disadvantages associated with exogenously introduced cytokines and hemopoietic factors.
A number of compounds are currently being evaluated that stimulate endogenous production of cytokines and hemopoietic factors in both experimental and clinical settings. There are universally known cases, including successful ones, of using microbial products for cancer therapy which in recent decades has been shown to be mediated via stimulation of the tumor necrosis factor endogenous production18. The products capable of evoking concomitant production of various cytokines and hemopoietic factors have presently come to be known as multi-cytokine inducers. Among these are a killed streptococcal preparation, Nocardia Opaca, and other bacterial products19,20,21. However, virtually all the substances possessing such capability are either killed microorganisms or microbial products or compounds having irregular composition, which results in their limited medicinal utility or even renders their therapeutic use impracticable. Thus, the problem of finding a medically and pharmaceutically acceptable inducer of the cytokine and hemopoietic factor endogenous production has not heretofore been resolved.
Oxidized glutathione (also known as glutathione disulfide and GSSG) will often be referred to as GSSG in this application.
GSSG is known as a dimmer of tripeptide glutathione (xcex3-glutamyl-cysteinyl-glycine) where two molecules of the tripeptide with the above structure are linked via a covalent disulfide bond between the cystamine residues. Therefore, both the tripeptide glutathione (glutathione, reduced glutathione, GSH; hereinafter referred to as GSH) and its dirmuer GSSG are natural metabolites present in animal tissues and biological fluids. At the same time, the natural blood level of GSSG is not sufficient for inducing the cytokine endogenous production in both normal and pathological conditions.
GSH is known to be one of the most important intermediates in the amino acid metabolism and a factor maintaining the intracellular homeostasis22,23. The reducing properties of GSH and its function as a donor of reduction equivalents, which is due to the sulfhydryl moiety of the cystamine residue, are of key importance. This characteristic of GSH is responsible for the substance playing a crucial part in one of the most important intracellular antioxidant systems, consisting of GSH as such and two enzymes of its reversible conversion into GSSG: glutathione peroxidase and glutathione reductase24,25. The permanent functioning of said system is essential for inactivating or reducing endogenously generated oxidants as well as active metabolites of foreign substances26,27.
GSH is also known to participate in detoxification reactions involving a group of enzymes collectively known as glutathione S-transferase28. These enzymes are capable of conjugating the GSH molecule with various xenobiotics by forming a bond between the latter and glutathione via the thiol moiety of the cystamine residue of the tripeptide. The subsequent degradation of the conjugate is catalyzed by the xcex3-glutamyl cycle enzymes, and may vary considerably depending upon the nature of the xenobiotic.
Under natural conditions, GSSG does not accumulate in amounts sufficient for inducing cytokine and hemopoietic factor production, due to a constant reduction of GSSG to GSH. The GSSG reduction to GSH also actively progresses in the intestines and liver upon GSSG oral administration, and as any product made of amino acids, the substance is proteolytically degradable in the gastrointestinal tract.
GSSG is known to be used as a components of a nutritional supplement utilized as an adjunct diet in treating patients29. However, being a peptide substance, most of the orally administered GSSG is digested in the gastrointestinal tract with the remainder being reduced in the intestinal and hepatic cells to GSH and not entering the circulation. Therefore, the delivery of GSSG into the organism through the gastrointestinal tract may eliminate the possibility of the realization of its activity as a stimulator of endogenous production of cytokines and hemopoietic factors.
An elevation of the GSH endogenous levels for medicinal utility is known to be suggested for boosting immunity30 and treating toxemias, poisonings, diabetes, mellitus, cardiovascular, infectious and other disorders31,32,33. Possible functions of GSH and GSSG appear in the literature.
Exogenous GSH or its direct (xcex3-glutamyl-cystamine, n-acetyl-cystamine, and n-acetyl-cystamine-glycine) or indirect (2-oxothiazolidine-4-carboxylate) biochemical precursors, or their salts and esters, are reportedly used as medicinal agents and dietary supplements in treating various diseases34,35,36,37,38.
GSH is also claimed to be useful as a chemoprotective agent that prevents neurotoxicity in cancer chemotherapy39 as well as in combination with antineoplastics in order to augment their effect40.
No reference, however, is currently available to GSSG as a medicine in its own right (sole substance) used to induce the endogenous production of cytokines and hemopoietic factors. The substance is known neither to have medicinal effects in human and animal diseases nor to be applied as a pharmaceutical agent for treating illnesses.
It is an object of the present invention to provide an active substance, and advantageous combinations of said substance and/or its derivatives with extenders and/or enhancers or modulators of its activity which are capable of inducing endogenous cytokine and hemopoietic factor production to an individual or a subject in need thereof.
xe2x80x9cSubject in need thereofxe2x80x9d as used in this application is intended to mean a mammal, e.g., man, domestic animals and livestock including cats, dogs, cattle and horses, having one or more manifestations of a disease in which stimulation of endogenous cytokine or hemopoietic factor (or both) production would be considered beneficial by those skilled in the art. xe2x80x9cTherapeutic agentxe2x80x9d as used in this application is meant to include any drug form of GSSG-containing material or GSSG alone, which has a therapeutic effect on neoplastic, infectious, hematologic, immunologic or other diseases. Therapeutic effect, as will be further defined, indicates any effect in man and other mammals which is beneficial, including curative, preventative, allowing maintenance at a beneficial level, or is in any way advantageous in connection with the body of man and other mammals.
In accordance with the present invention, it is GSSG that upon parenteral administration induces the endogenous cytokine and/or hematopoietic factor production in an individual or subject in need thereof, in both health and disease.
Having performed studies in search for a medically and pharmaceutically acceptable inducer of the cytokine and hemopoietic factor endogenous production, the applicants discovered a new property of a previously known substance, oxidized glutathione (oxidized glutathione, glutathione disulfide, GSSG; hereinafter often referred to as GSSG).
Being administered parenterally or acting on isolated cells, the substance is capable of inducing production of several cytokines and hemopoietic factors in mammals (animals and humans) in both health and disease.
The inducer or stimulator of the endogenous cytokine and hemopoietic factor production is oxidized glutathione (GSSG) which is a dimmer of reduced glutathione having the structure xcex3-glutamyl-cysteinyl-glycine, where the two molecules of the tripeptide are linked via a covalent disulfide bond between the cystamine residues.
According to the invention, a method is provided for stimulating endogenous production of cytokine and hemopoietic factors by introducing to a mammalian body in need of stimulation of cytokine or hemopoietic factor or both, an effective amount of oxidized glutathione for a sufficient period of time to stimulate said endogenous production to obtain a therapeutic effect.
Preferably, the glutathione is introduced parenterally or topically. In a preferred form, the method is carried out by introducing the oxidized glutathione (GSSG) or its derivatives with an extender of half life and/or enhancers or modulators to enhance the desired effect of stimulating endogenous production of cytokines and hemopoietic factors and producing a therapeutic effect in a body.
Preferably, the GSSG derivative is selected from the group of compounds representing a molecule of GSSG chemically modified by binding covalently as for example: with cysteamine-(2-mercaptoethylamine), lipoic acid (6,8-thioctic acid), camosine (b-alanyl-hystidine), adenosine (9-xcex2-D-ribofuranosyladenine), methionine (2-amino-4-[methylthio]butanoic acid); and both the D and L forms of the amino acids set forth in this paragraph can be used.
Particularly desirable derivatives are GSSG covalently bound either to cysteamine (S-thioethylamine-glutathione disulfide), or to lipoic acid (bis-[6,8-thiooktanil]xe2x80xa2glutathione disulfide), or to camosine ([b-alanyl-hystidil]xe2x80xa2glutathione disulfide), or to adenosine ([9xcex2-D-ribofuranosyladenil]xe2x80xa2glutathione disulfide), or to methionine (bis-[2-amino-4-[methylthio]butanoil]xe2x80xa2glutathione disulfide), or mixtures thereof and including the D and/or L forms of amino acids herein.
Preferably, the extender is selected from the group consisting of pharmaceutically acceptable pro-oxidant compounds, (hydrogen peroxide, ascorbic acid) compounds capable of forming both weak ionic and coordinating links which stabilize molecule of GSSG (dimethyl sulfoxide), or materials which are competitors of NADP-H-dependent reduction of GSSG into GSH catalyzed by glutathione reductase, compounds capable of producing reversible inhibition of reduction of NADP+ into NADP-H catalyzed by glucose-6-phosphate-dehydrogenase or by other NADP-H-dependent enzymes, or mixtures thereof.
Particularly desirable extenders are hydrogen peroxide, inosine, ascorbic acid, dimethyl sulfoxide, or cystamine or mixtures thereof.
Preferably, the enhancer/modulator is selected from the group consisting of methyl moiety donators (such as choline-chloride{[2-hydroxyethyl]trimethylammonium chloride} or S-adenosyl-methionine), representatives of intracellular redox-oxidative pairs (such as lipoic/dehydrolipoic, folic/dehydrofolic, ascorbic/dehydroascorbic acids). An enhancer or modulator or enhancer/modulator as used herein is meant to be a material which increases or changes beneficially in terms of curative outcomes the therapeutic effect of GSSG or its derivatives, but is not an extender of half life of the GSSG.
Particularly desirable enhancers or modulators are choline-chloride, S-adenosylmethionine, lipoic (6,8-thioctic) and folic (pteroylglutamic) acids.
In the preferred form, GSSG is introduced to the body at a dose of from 0.01 to 0.5 mg of GSSG base per kg of body weight for GSSG base and its salts, and from 0.01 to 1.0 mg for GSSG derivatives, at least one time during each 24 hour period, although it can be continuously injected or otherwise introduced to the body to have a 24 hour total dosage of from 0.01 to 0.5 mg per kg of body weight for GSSG base and its salts, and from 0.01 to 1.0 mg for GSSG derivatives each 24 hour period. Preferably, administration and introduction to the body is carried out until a desired stimulating effect increasing production of cytokines and hemopoietic factors and providing a therapeutic effect is obtained.
According to the invention, a therapeutic agent for treating neoplastic, infectious, hematologic, immunologic and other diseases is provided, comprising an effective amount of oxidized glutathione, along with a pharmaceutically acceptable excipient. Preferably, the oxidized glutathione for parenteral use is in a pharmaceutically acceptable solvent as, for example, an aqueous solution including water, glucose solution, isotonic solutions of sodium chloride, buffered salt solutions. Preferably, a pharmaceutically acceptable extender capable of enhancing and prolonging therapeutic effect as by increasing the half life of oxidized glutathione; or a pharmaceutically acceptable enhancer or modulator of GSSG activity by mechanisms other than increasing the GSSG half life, is used along with the GSSG.
The applicants have for the first time shown that an immediate action of exogenous GSSG or its salts on mammalian (human and laboratory animal) cells capable of producing cytokines and hemopoietic factors, exerts stimulation on the synthesis of these molecules and their increased level in the blood serum (in vivo conditions) or culture media (ex vivo or in vitro conditions). The method suggested can bring about the effect of stimulating production of cytokines and hemopoietic factors, and this effect is elicited by the administration of GSSG into the organism or entering into the cultural media, as well as by the administration of GSSG in combination with pharmacologically active formulations mediating either the prolongation of glutathione""s retaining the oxidized form or enhancing or beneficially modulating its activity. The studies performed by the applicants have revealed GSSG and its formulations to possess a therapeutic effect in various experimental and clinical pathological conditions.
The revealed GSSG-induced stimulation of the endogenous cytokine and hemopoietic factor production in the body results in antitumor, anti-infective, hemopoietic, immunomodulatory and other pharmacological effects resulting, in turn, to a greater or lesser extent therapeutic or preventive effect in various diseases.